Project summary Human mesenchymal stem cells (hMSCs) are key players in wound healing. In the wound, hMSCs regulate in?ammation by decreasing pro-in?ammatory cytokines and increasing anti-in?ammatory cytokines. hMSCs aid in repair and regeneration of damaged tissue by coordinating the local cell response using cell-cell communi- cation. Since hMSCs are integral to wound healing, implantable synthetic hMSC-laden scaffolds are actively being developed to deliver additional cells to wounded tissue. Current in vitro studies engineer cues into the scaffold microenvironment to enhance cell delivery. However, these studies do not account for additional cues in the native wound environment. hMSCs migrate to wounds by chemotaxis towards chemical signals released by the wounded tissue. These signals include cytokines, chemokines and growth factors. hMSCs encapsulated in a scaffold during implantation will also receive chemical cues from the wound, which, once understood, can be leveraged to design materials that manipulate and direct hMSCs to wounds. We propose to quantitatively characterize hMSC motility and hMSC-mediated pericellular degradation within a poly(ethylene glycol)(PEG)- peptide hydrogel scaffold in response to chemical cues presented in the environment and locally tethered into the scaffold. This versatile hydrogel is designed to be enzymatically degraded by cell-secreted matrix metallopro- teinases (MMPs). This work will use cell tracking to quantify hMSC speed and persistence. We will characterize cell-mediated pericellular degradation using multiple particle tracking microrheology (MPT). This technique char- acterizes spatio-temporal changes in rheological properties by measuring the Brownian motion of embedded particles. Our previous work has determined that pericellular degradation and cell speed are related. We hy- pothesize that native environmental cues can be used in conjunction with material design to direct cell motility. Because hMSC behavior will be changed in the presence of cytokines in the environment, the goal of the pro- posed work is to characterize the relation between pericellular degradation and hMSC motility in response to locally and environmentally presented cytokines to establish design rules to enhance cell delivery to wounded tissue. Speci?c Aim 1 will determine the change in hMSC motility and pericellular degradation in response to cytokines in the environment. Cell-laden hydrogels will be incubated in media with cytokines. Cell tracking and MPT will characterize changes in hMSC migration and the cell-mediated scaffold degradation pro?le. Speci?c Aim 2 will determine if a local cytokine gradient can direct hMSC motility when cytokines are also present in the environment. We will tether gradients of thiol-modi?ed cytokines into the hydrogel to direct hMSC motility. The effectiveness of the cytokine gradient will be measured when the scaffold is incubated in media with cytokines, mimicking aspects of the wound environment. This work will establish design rules that leverage native environ- mental and locally presented chemical cues to increase and direct hMSC motility to enhance deliver of hMSCs to a wound.